8月2日，華中農(nóng)業(yè)大學(xué)水產(chǎn)學(xué)院高澤霞教授團隊聯(lián)合中國科學(xué)院南海海洋研究所林強研究員團隊在魚類肌間刺方面的研究成果以“Single-cell transcriptomes and runx2b?/? mutants reveal the genetic signatures of intermuscular bone formation in zebrafish”為題在National Science Review期刊發(fā)表。研究明確了魚類肌間刺形成的關(guān)鍵細(xì)胞群，鑒定到調(diào)控魚類肌間刺發(fā)育的關(guān)鍵基因runx2b，揭示了runx2b調(diào)控魚類肌間刺形成的分子機制。

 

　　世界主要養(yǎng)殖魚類都含有一定數(shù)量的肌間刺，特別是我國主養(yǎng)的青草鰱鳙鯉鯽魴等大宗淡水魚類，食用時極易出現(xiàn)卡喉嚨的危險，嚴(yán)重影響了其產(chǎn)品的經(jīng)濟價值。高澤霞教授團隊前期研究已利用轉(zhuǎn)錄組、蛋白組、小RNA組、全轉(zhuǎn)錄組等組學(xué)構(gòu)建了魚類肌間刺發(fā)生發(fā)育的組學(xué)資源庫，并篩選到了關(guān)鍵的信號通路和候選功能基因，但肌間刺形成的關(guān)鍵調(diào)控基因尚不明確。

 

　　該研究首先利用單細(xì)胞轉(zhuǎn)錄組測序技術(shù)（scRNA-seq）定義了野生型斑馬魚肌間刺所在區(qū)域的18個細(xì)胞類群，篩選到了肌間刺形成顯著相關(guān)的肌腱祖細(xì)胞、肌腱分化細(xì)胞、成熟肌腱細(xì)胞和成骨細(xì)胞類群。細(xì)胞擬時序分析結(jié)果推測肌間刺是由肌腱祖細(xì)胞經(jīng)肌腱分化細(xì)胞，再分化為成骨細(xì)胞而形成的。根據(jù)基因在細(xì)胞軌跡上的表達(dá)模式，篩選到10個與肌間刺形成密切相關(guān)的功能基因，并在斑馬魚上運用CRISPR-Cas9基因編輯技術(shù)分別構(gòu)建了基因突變品系。最終發(fā)現(xiàn)runx2b突變斑馬魚肌間刺完全缺失，且其個體生長、其它骨骼單元形成、肌肉脂肪酸和氨基酸含量等性狀均未受到顯著影響。scRNA-seq分析同時揭示了runx2b突變體斑馬魚的肌間刺發(fā)生區(qū)成骨細(xì)胞數(shù)量的顯著減少，在個體和細(xì)胞水平明確了TGF-β/BMP signaling pathway在肌間刺形成過程中的重要調(diào)控作用。

 

　　此外，該研究團隊在經(jīng)濟魚類團頭魴（武昌魚）上運用CRISPR-Cas9基因編輯技術(shù)構(gòu)建了runx2b基因F0代突變體，發(fā)現(xiàn)runx2b突變體團頭魴的肌間刺發(fā)育亦受到顯著影響，部分個體肌間刺數(shù)目減少30%以上，表明runx2b基因?qū)?jīng)濟魚類肌間刺形成亦具有重要調(diào)控作用。鑒于團頭魴性成熟時間為2年，團隊擬在后續(xù)研究中繁育獲得純和突變品系，以期獲得肌間刺完全缺失的團頭魴新種質(zhì)。

 

　　華中農(nóng)業(yè)大學(xué)水產(chǎn)學(xué)院博士后聶春紅、副教授萬世明、博士生陳宇龍為論文共同第一作者，水產(chǎn)學(xué)院高澤霞教授和中國科學(xué)院南海海洋研究所林強研究員為論文共同通訊作者，水產(chǎn)學(xué)院王衛(wèi)民教授參與指導(dǎo)了本研究，比利時根特大學(xué)Paul Witten教授與Ann Huysseune教授，巴西莫吉達(dá)斯克魯茲大學(xué)Alexandre Hilsdorf教授等參與了本項研究。該研究得到國家大宗淡水魚類產(chǎn)業(yè)技術(shù)體系、湖北洪山實驗室、國家自然科學(xué)基金、中國科學(xué)院前沿科學(xué)重點研究等項目的支持。

 

　　英文摘要：

 

　　Intermuscular bones （IBs） are mineralized spicules, present in the myosepta of many, but not all teleost species. IBs are often small and sharp, and they consequently limit how the fish can be processed, cause injury or trauma if lodged in consumers' throat or mouth and therefore affect the appeal of the fish to many consumers. The development of IBs in teleosts is still not fully understood and the molecular basis of IB development remains to be established. Here, the characteristics of IB tissue is evaluated based on single-cell transcriptomic in wild-type zebrafish. The analysis defined 18 distinct cell types. Differentiation trajectories showed that IBs are derived from tendons and that a core tendon-osteoblast cell lineage is related to IB formation. In particular, the functions of 10 candidate genes were evaluated via CRISPR-Cas9 mutants. Among those, runx2b?/?mutants completely lost IBs, while swimming performance, growth, and bone mineral density were not significantly different form runx2b+/+ zebrafish. Comparative scRNA-seq analysis in runx2b?/? and runx2b+/+ zebrafish revealed the role of osteoblasts in IB formation. In addition, differentially expressed genes were enriched in TGF-β/BMP pathway after runx2b deletion. This study provide evidence for a crucial role of runx2b regulation in IB formation. Genetic breeding can target runx2b regulation and generate strains of commercial fish species without IBs, which can improve the safe consumption and economic value of many farmed fish species.

 

　　論文鏈接：https://doi.org/10.1093/nsr/nwac152